Phosphorus is an essential element for all organisms. Plant-derived feeds used for the production of domestic animals contain phosphorus, 50 to 70% of which is present as phytic acid. Phytic acid is a major storage substance of phosphoric acid, existing in a large amount in plant seeds. However, phytic acid is excreted without digestion and absorption in the digestive organs in single-stomach animals such as pigs, chickens, etc. That is, phytic acid is a storage substance of phosphoric acid, but its phosphorus is not utilized at all. Accordingly, phosphoric acid is added to feed for single-stomach animals for the purpose of growth promotion.
Addition of phosphoric acid to the feed leads to an increase in the amount of phosphorus in excrement. In recent years, excrement from domestic animals increase considerably as the production of domestic animals increases more and more, whereby an environmental problem is now caused in the world. In particular, phosphorus contained in excrement is mentioned as a factor causing the phenomenon of nutrition enrichment in lakes and marshes, so the amount of phosphorus in excrement comes to be regulated, and there arises necessity for countermeasure.
In addition to the problem of excreted phosphorus, phytic acid chelates divalent metals important as a nutritive source, such as magnesium, calcium, zinc and iron, thereby making its absorption into animals difficult and reducing the nutritive value of feed. Accordingly, phytic acid is regarded as an anti-trophic factor.
From the foregoing, it has been examined to decrease the amount of phosphorus in excrement by treating the feed with a phytase known widely as an enzyme capable of hydrolyzing a salt of phytic acid into inositol and inorganic phosphoric acid in order to utilize phosphoric acid released from phytic acid in place of phosphoric acid conventionally added in feed, and it has also been examined to improve the nutritive value of the feed by decomposing phytic acid as an anti-trophic factor [U.S. Pat. No. 3,297,548 (1967); J. Nutrition, 101, 1289-1294 (1971)].
Known as phytase-producing microorganisms are bacteria such as Bacillus subtilis and Pseudomonas, yeasts such as Saccharomyces cerevisiae, and filamentous fungi such as Aspergillus terreus, Aspergillus ficcum and Aspergillus awamori.
For phytase derived from Aspergillus ficcum, its purification and biochemical properties are described in Preparative Biochem., 18, 443-458 (1988), and its gene and amino acid sequence are described in Gene, 127, 87-94 (1993).
For phytase derived from Aspergillus awamori, its nucleotide sequence and amino acid sequence are described in Gene, 133, 55-62 (1993).
Michaelis constants (Km) for phytases known so far are 0.57 mM for wheat bran-derived phytase [Agr. Biol. Chem., 26, 794-803 (1962)], 0.17 mM for rice bran-derived phytase [Agr. Biol. Chem., 53, 1475-1483 (1898)], 117 .mu.M for maize (Zea mays)-derived phytase, 250 .mu.M for Aspergillus ficcum-derived phytase (WO 91/05053), 330 .mu.M for Aspergillus oryzae-derived phytase, 150 .mu.M for Bacillus subtilis-derived phytase, 500 .mu.M for Bacillus natto-derived phytase, and 130 .mu.M for Escherichia coli-derived phytase.
To demonstrate the performance of the enzyme, the concentration of a substrate is necessary to be higher than Km, and if an enzyme with low Km and an enzyme with high Km have the same maximum reaction rate (Vmax), the enzyme with low Km does not decrease a reaction rate even at a lower substrate concentration as compared with the enzyme with high Km.
That is, when compared with the enzyme with high Km, the enzyme with low Km is advantageous in that a sufficient degradation rate can be achieved even at a lower substrate concentration, thereby minimizing the amount of the remaining substrate.
Accordingly, there is a demand for an inexpensive phytase with a low Km value for phytic acid, which phytase degrades phytic acid being an anti-trophic factor contained in feed, thereby improving the nutritive value of the feed and simultaneously achieving an efficient utilization of phosphoric acid released by the degradation.